A 66 kD protein has recently been shown to be a novel subunit of mammalian neurofilaments. It is distinct from all known neurofilament subunits. The goal of project is to explore the expression of this protein in development, specifically in the granule cells of the cerebellum . A polyclonal antibody, specific to the 66 kD NF protein will be used to demonstrate, by immunohistochemical staining, the appearance of neurofilaments in the cerebellum of paranatal rat. Correlations will be made between the expression of neurofilaments and the maturation of the granule cells. Granule cells from postnatal cerebella will be established in culture and used as an in vitro model to study neurofilament expression. The relationship of the soluble and the insoluble pools of the 66 kD NF protein will be explored by in vitro radiolabeling. Existence of intermediate filaments in the axons of granule cells, which until recently were not thought possible, will now be more carefully evaluated with both electron microscopy and immuno-EM. A cDNA probe will be constructed specifically to the 66 kD protein by the polymerase-chain reaction method. In addition, expression libraries of rat brain DNA will be screened with either antibodies or synthetic oligonucleotides. The cDNA probe will be used to sequence the gene encoding the 66 kDa NF protein, allowing the unambiguous assignment of that protein to a subtype of intermediate filaments. In situ hybridization of the cDNA probe will complement the data on the developmental expression of NFs as obtained from immunocytochemical stainings. The appearance of mRNA that encodes the 66 kD NF protein during fetal development will be detected by polymerase chain reaction.